Established cell line of microglia

ABSTRACT

A subcultivatable, established microglia having the following properties. (a) Form: Both or either of a macrophage-like or spherical form in the presence of a granulocyte-macrophage colony stimulation factor and a branched form similar to branched microglia present in the brain in the absence of the factor. (b) Functional characteristics: specific affinity for the brain highly poor phagocytic action. (c) Cell proliferation: proliferative depending upon a granulocyte-macrophage colony stimulation factor. Preparation, separation, and screening methods of the microglia, a pharmaceutical composition using the microglia, and a method for treatment of cerebral diseases using the composition.

TECHNICAL FIELD

The present invention relates to a subcultivatable, established cellline of microglia, a method of separating the same, and use thereof as apharmaceutical carrier.

Further, the present invention relates to a microglia into which anextraneous gene or a drug was introduced, a method of introducing it,and a pharmaceutical composition comprising the same.

BACKGROUND ART

There are quite a number of hereditary diseases in the nervous system,which occur due to various causative factors, for example by defect of asingle enzyme, etc. or by unknown reasons. Under these circumstances,supplementary therapy is used to cope with a large number of suchdiseases.

A large-number of studies have been made worldwide on the system ofselective delivery to the brain. For introducing a gene into the brainin an animal, a method of using neuronphilic viruses as adenovirusvectors is devised, and a system for introducing a gene specificallyinto neurons is known (Kozarsky, K. F and Wilson, J. M., Curr. Opin.Genet. Dev. 3, 499-503, 1993). A method of using a retrovirus vector isalso devised and has succeeded in introducing a gene into hepatic cells,bloodcells, etc. (Mullingan, R. C., Science 260, 926-932, 1993).

In the brain, however, the blood-brain barrier is present, so it isdifficult to conduct supplementary therapy and to introduce an effectivedrug, and even if a substance (e.g. anticancer drug, DNA, etc.) isintroduced from a peripheral position, it cannot be introducedspecifically into the brain. Therefore, there was no method other thandirect injection of the substance by surgical operations.

As a method not involving invasive means such as surgical operations,there is a method of utilizing liposomes, and liposomes rendered capableof introduction into the brain relatively easily by changing theirconstitutional elements were developed by a Japanese group. However,even in this method too, incorporation of liposomes into the brain is aslow as about 1% based on the injection amount, so this method cannot besaid to be specific for the brain.

As described above, the blood-brain barrier is present in the brain, sothe brain is almost free of infiltration with cells or a substance fromthe periphery, thus making it difficult to introduce a drug or a geneinto the brain. Actually, infiltration of the normal brain withimmunocytes such as T cells and macrophages is hardly observed.

Microglia are cells with macrophage-like properties in the centralnervous system, which not only function as immunocompetent cells ininflammatory reaction and viral infection and as phagocytes for removingcells but also play a central role in a cytokine network in the centralnervous system (Sawada, M. et al., Int. J. Dev. Neurosci., 13, 253-264,1995). Recently, microglia have been revealed to be essential forexpression of high-level brain functions such as learning andmemorization and considered to be specialized cells having a rolespecific for the brain. Up to now, it has been considered that microgliaoriginate from monocytes having infiltrated the brain in the perinatalperiod and are specialized and differentiated.

The microglia can be obtained by primary culture of brain cells. Forthis primary culture, however, the brain should be excised and purifiedfor use, and primary culture usually requires a period of about 2 weeks,so the procedures are cumbersome. Further, the cells are difficult toproliferate during culture and hard to subculture, so after primaryculture, it is extremely difficult to introduce a gene into themicroglia to express it therein.

In a process of studies on microglia considered as brain-specificmacrophage, the present inventors have succeeded in obtaining highlypurified microglia and examined the properties, and as a result, wefound that unlike the macrophage, microglia have specific affinity forthe rain.

Further, the inventors found that microglia are determinativelydifferent from macrophage in respect of affinity for the brain and theability to infiltrate the brain. Further, the inventors examined thedistribution thereof by staining in a method capable of distinguishingthe two, and found that microglia are present from an early developmentstage in the brain. Accordingly, it is considered that microglia are notderived from a monocyte differentiated and matured in the bone marrow,but are derived from a cell group which have a specific affinity for thebrain, and infiltrate the brain at an early development stage and cometo regulate high-level functions such as cerebral morphogenesis andlearning and memorization.

Accordingly, the inventors injected an isolated macrophage and microgliainto rat peripheral arteries to compare the two for selective affinityfor the brain, and as a result we found that when the microglia labeledwith a fluorescent pigment was injected, many fluorescent cells wereobserved in the brain but hardly observed in the liver. On the otherhand, when the macrophage was injected, fluorescent cells were hardlyobserved in the normal cell, but many fluorescent cells were observed inthe liver.

Then, the inventors examined whether a cell strain of microgliaestablished by the inventors can express a gene selectively in the brainby introducing a lacZ expression vector into the cell strain andinjecting the resultant cells into a rat blood stream, and as a result,the inventors could detect the activity of β-galactosidase in a sectionof the rat brain into which the lacZ-expressing cells had been injected.From these results, it was found that the microglia unlike themacrophage is a cell having specific affinity for the brain, and byutilizing this affinity, a specific substance or gene can be introducedvia a peripheral blood stream into the brain.

DISCLOSURE OF INVENTION

That is, the present invention relates to a subcultivatable establishedmicroglia and in particular to an established cell line of microgliahaving the following properties:

(a) form: having a macrophage-like or globular form in the presence ofgranulocyte-macrophage colony-stimulating factor, and in the absence ofsaid factor, a branched form similar to branched microglia present inthe brain, or both of the above forms;

(b) functional characteristics: having specific affinity for the brain,and having a strong phagocytic ability; and

(c) cell growth ability: growing depending on granulocyte-macrophagecolony-stimulating factor.

Further, the present invention relates to a method of separating asubcultivatable established cell line of microglia from microglia cellsin the presence of a cytokine, preferably in the presence ofcolony-stimulating factor (CSF), more preferably in the presence ofgranulocyte-macrophage colony-stimulating factor (GM-CSF), still morepreferably in the presence of IL-3 and/or purified astrocytes.

Further, the present invention relates to a pharmaceutical carriercomprising the established cell line of microglia described above.

Further, the present invention relates to the above-descried establishedcell line of microglia having a gene or a drug introduced into it.

Further, the present invention relates to a pharmaceutical compositioncomprising the above-described microglia having a gene or a drugintroduced into it and a pharmaceutical carrier and in particular to apharmaceutical composition which is an agent for treatment of cerebraldiseases.

Further, the present invention relates to a method for screening orproducing a microglia having an extraneous gene introduced into it,comprising introduction of an extraneous gene and a gene expressing afluorescent protein, preferably a fluorescent protein derived from ajellyfish, into a microglia. As the microglia in the method of thepresent invention, the conventional microglia can also be used, but theabove-described established cell line of microglia of the presentinvention is preferably used.

Further, the present invention relates to a method of treating cerebraldiseases, which comprises using the above-described pharmaceuticalcomposition to deliver a drug or gene specifically to the brain.

Hereinafter, the present invention is described in more detail.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a photograph showing the form of the established cell line ofmicroglia of the present invention (morphology of the cells).

FIG. 2 is a photograph showing the difference in tissue specificitybetween the established cell line of microglia of the present inventionand macrophages (morphology of the cells).

FIG. 3 shows electrophoresis indicating cytokine expression bystimulation with lipopolysaccharides.

FIG. 4 shows GM-CSF-dependent proliferation of the established cell lineof microglia of the present invention.

FIG. 5 is a photograph indicating gene expression in a rat brain(morphology of the cells).

FIG. 6 shows gene expression in a rat brain.

FIG. 7 shows a result of FACS analysis of cells into which a gene wasnot introduced.

FIG. 8 shows a result of FACS analysis of cells into which GFP wasintroduced.

BEST MODE FOR CARRYING OUT THE INVENTION

The established cell line of microglia of the present invention can bepurified from mouse or rat brain cells after primary culture and thenseparated by the following means from this purified microglia. Inaddition, because the established cell line of microglia of the presentinvention is easily handled and has affinity for the brain, a gene ordrug can be introduced into the established cell line of microglia andinjected into peripheral blood vessels to express the gene in the brainor to deliver the drug to the brain specifically.

Hereinafter, the process for producing the established cell line ofmicroglia of the present invention is described in more detail.

(1) Purification of Microglia

First, the meninges are removed from collected mouse or rat brains anddivided into single cells by use of a pipette, nylon mesh, etc. Themouse and rat are preferably newborn. The mouse includes, but is notlimited to, C57BL6, C3H, ICR, Balb/c, etc., and the rat includes, but isnot limited to, Fisher, Wister, SD, etc.

The resulting cells are plated on a usual animal cell culture medium(e.g. EMEM containing 10% FCS or CS) and cultured for 10 to 14 days. Themedium is exchanged with fresh one every 3 to 4 days.

Then, the cultured cells thus obtained are selected to prepare anestablished cell line in the subsequent step.

There are microglia called type I and type II, and type I is floatingcells which are removed from the culture vessel upon mechanicalstimulation of cells after primary culture (that is, by splashing thecells with a medium through a pipette, by shaking the culture vessel,etc.). Type II is cells (adherent cells) not floated by said mechanicalstimulation. Since the microglia of the present invention belongs totype II, purified microglia can be selected in the following manner.

The adherent cells which do not float by the above mechanicalstimulation are treated with trypsin-EDTA , then divided into singlecells, plated on a non-treated plastic dish (non-coat plastic vessel),and allowed to adhere to it. Generally, a conventional culture vessel istreated with chemicals so as to be positively charged, but a dish notsubjected to this treatment should be used in order to obtain theadherent cells. After incubation in a CO₂ incubator at 37° C. for 1hour, cells floating in the medium after mechanical stimulation areremoved, and cells capable of proliferation on the vessel are recoveredby Rubber Policeman, etc., and the same procedure is repeated twice toobtain cells to be used in establishing a cell line of microglia.Although the purified microglia thus obtained is of adequate purity,cell sorter, etc. can also be used to further improve purity.

(2) Separation of Established Cell Line of Microglia

To separate the subcultivatable established cell line of microglia ofthe present invention from the purified microglia obtained in the mannerdescribed in (1) above, the purified microglia is cloned through culturein the presence of a cytokine, preferably colony-stimulating factor(CSF), more preferably granulocyte-macrophage colony-stimulating factor(GM-CSF). As the cytokine allowed to be present during culture, GM-CSFmay be used singly, but IL-3 and/or a supernatant derived from purifiedastrocytes may be allowed to be present in addition to GM-CSF. Thecytokine used may be the naturally occurring orgenetic recombinant one.Specific procedures are for example as follows:

The purified microglia obtained in (1) above is plated on a vessel ofabout 10 cm diameter and cultured for 7 to 10 days in the presence ofgenetic recombinant granulocyte-macrophage colony-stimulating factor(rGM-CSF). After culture, the cells are recovered, and using limitingdilution, they are further cultured for 4 to 10 weeks in the presence ofrGM-CSF. Then, cells having formed a single colony in each well in thetest plate are released by Rubber Policeman whereby the cloned cells aresorted and separated to finally give an established cell line ofmicroglia.

The established cell line of microglia thus obtained has the followingproperties.

(a) Form

When observed after staining with a fluorescent pigment under afluorescence microscope or without staining under a phase contractmicroscope, the cells have a macrophage-like or globular form in thepresence of rGM-CSF, or in the absence of rGM-CSF, a branched formsimilar to branched microglia present in the brain. Otherwise the cellshave both of the above forms.

(b) Functional Characteristics

Upon administration into an artery of a mouse, the established microgliaof the present invention moves specifically to the brain, indicatingthat it has specific affinity for the brain. Further, when stimulationis given with lipopolysaccharides the microglia produces interleukin-1(IL-1) and interleukin-6 (IL-6). Upon stimulation with interferon γ, itproduces IL-5. This property is different from that of macrophagesbecause macrophages do not express IL-5 upon stimulation with IFN-γ.When the phagocytic ability of the cell line of the present invention isexamined using incorporation of a fluorescent pigment as an indicator,it has a strong phagocytic ability. The established cell line ofmicroglia of the present invention has a phagocytic ability which ishundreds to thousands times higher than that of astrocytes.

(c) Cell Growth Ability

Because the established microglia of the present invention is notproliferated after removal of rGM-CSF from the medium, it isproliferated depending on GM-CSF.

The form and characteristics of the established microglia of the presentinvention are exemplified in specific examples in FIGS. 1 to 4.

(3) Introduction of Gene into the Established Cell Line of Microglia

Introduction of a gene into the established cell line of microglia ofthe present invention is important for expressing the gene specificallyin the brain. A desired gene can be obtained by known cloning means, andany commercially available gene can also be used, and the type of geneis particularly not limited.

As a means of efficiently introducing a gene into the established cellline of microglia, there is for example a method of using DOTAP(Boehringer-Mannheim Co.). That is, there is a method of culturing theestablished cell line of microglia together with a desired gene in agene-introducing medium containing DOTAP. They are cultured in a CO₂incubator at 37° C. for 16 to 24 hours and further cultured togetherwith rGM-CSF for 30 to 72 hours (preferably for 48 hours) in a mediumfor culturing microglia.

In addition to the above-described means, the method of introducing thegene into the microglia includes conventional means such as calciumphosphate method, DEAE dextran method, lipofection method,electroporation method, particle gum method, etc.

Out of known methods of separating a strain stably expressing anintroduced gene, the most generally used method is a method wherein adrug resistance gene is introduced simultaneously with a desired geneinto cells and expressed therein, and cells other than those cellscoming to express the gene stably and constantly by incorporating thegene into their chromosomal DNA are perished by chemical treatment andexcluded from the culture, whereby the desired cells are separated.

When this screening method is applied to the microglia, the microgliacoming to express the introduced gene stably and constantly recognizedead cells thus expressing a strong phagocytic ability andsimultaneously undergoing activation to lose their cell growth ability.Accordingly, the method of introducing a gene in the present inventionis preferably a method wherein an expression vector modified so as to becapable of expressing a fluorescent protein, preferably green florescentprotein (GFP) derived from an aurelia, in higher animals is used inplace of a drug resistance gene, and by using a difference influorescence intensity, the microglia is separated as the desired cellsexhibiting stable and constant expression.

Whether the cells having the gene introduced into them have reached thebrain and whether the gene has been expressed can be confirmed in thefollowing manner: The cells to be introduced are stained with afluorescent pigment specific for phagocytes. After introduction of thecells into an animal, the brain is excised and frozen, from which asection of about 8 microns in thickness is prepared and examined forfluorescent cells under a fluorescence microscope, or the section isactivity-stained with a substrate for the introduced gene.

The cells can also be confirmed using magnetic resonance image (MRI),positron emission tomography (PET), etc. For example, contrast media,etc. for MRI may be incorporated into the cells which are then injectedinto an animal so that the cells can be monitored in the animal.According to these methods, it is not necessary to kill the animal, andthe cells can be monitored easily in a non-invasive manner.

EXAMPLES

Hereinafter, the present invention is described in more detail byreference to Examples. However, the scope of the present invention isnot limited to the Examples.

Example 1

Separation of Established Clone of Microglia

(1) Isolation of Microglia

Brains were excised from newborn mice (C57BL6, op/op) and newborn rats(Fisher), and meninges were removed in an ice-cold microglia culturemedium (referred to as Mi medium; Eagle's MEM containing 10% bovineserum, 0.2% glucose and 5 μg/ml bovine insulin). The cells of meningeswere divided into single cells with a Pasteur pipette or nylon mesh andthen cultured in Mi medium. For the cells from the mouse brains, 20 mlMi medium was used per one brain, and for the cells from the rat brains,40 ml Mi medium was used per one brain, and the former cells wereincubated in 2 culture vessels of 10 cm diameter and the latter cells in4 culture vessels of 10 cm diameter in a CO₂ incubator (5% CO₂, 95% air)at 37° C. for 10 to 14 days. The medium was exchanged with fresh oneevery 3 to 4 days.

When phase-bright round cells (PBRCs) appeared, the PBRCs were removedby mechanical shaking, and the remaining cells were removed with 200U/ml trypsin-0.02% EDTA and incubated in a non-coat plastic vessel at37° C. for 30 minutes. The cells which adhered to the non-coat plasticvessel were washed twice with Mi medium, and the cells were then removedand recovered. The same procedure was repeated further twice to givepurified microglia.

(2) Separation of Clones

1×10⁵ purified microglia cells obtained in (1) above were plated on a 10cm vessel and cultured for 7 days in Mi medium in the presence ofrGM-CSF (Genzyme Co.).

The cells were recovered and counted, and clones were obtained from thecells in the following manner using limiting dilution. The cells wereput to each well on a 96-well plate (Falcon Co.) at a density of 0.5cell/well (in 100 μl) and cultured for about 3 weeks in the presence of2 ng/ml mouse gene recombinant GM-CSF (Genzyme Co.). Each well wasexamined for the presence of the clone, and the target clones wereseparated.

As a result, five kinds of established microglia (Ra2,GMI-M6-1,GMI-M6-3, GMI-M5-2, GMI-MF11) derived from the mouse brains andone kind (GMI-R1) from the rat brains were obtained.

Among these, the established cell lines of microglia, Ra2 and GMI-R1,have been designated as “mouse microglia Ra2” and “rat microglia GMI-R1”and deposited as FERM P-16109 and FERM P-16110, respectively, with theNational Institute of Bioscience and Human-Technology, Agency ofIndustrial Science and Technology, Japan.

The properties of the resulting clones were then examined.

(a) Form

When GMI-RI incubated in the presence or absence of rGM-CSF was observedunder a phase contrast microscope, it had a macrophage-like or globularform in the presence of rGM-CSF (FIG. 1A) and a branched form in theabsence of rGM-CSF (FIG. 1B).

(b-1) Functional Characteristics (affinity for the brain)

The established cell line of microglia GMI-R1 of the present inventionwas allowed to adhere to a plastic vessel. Separately, a fluorescentpigment PKH26 (Zynaxis Co.) prepared in a phagocyte-staining solution(Diluent B, Zynaxis Co.) and 10 % serum were mixed at a ratio of 1:1 andadded to the above vessel, and GMI-RI was stained with the fluorescentpigment at 37° C. for 15 minutes (Ishihara, S., Sawada, M. et al., Exp.Neurol., 124, 219-230, 1993).

The cells were recovered and 2×10⁶ cells were injected into an artery inthe armpit of each of 5-week-old rats from the same strain (Fisher). 48hours and 1, 2, and 3 weeks after injection, each organ was excised fromthe rats and frozen in a solution of OCT (Tissue Tek Co.).

Microglia GMI-R1, and macrophages which was isolated for comparison fromthe abdomen of a rat of the same strain (Fisher) by washing it with coldPBS, were labeled respectively with a fluorescent pigment specific forphagocytes and then injected into armpit arteries of rats, and tissuesections were prepared for examination of the tissue orientation.

After the established cell line of microglia of the present inventionwas injected, many fluorescent cells were observed in normal brain cells(FIG. 2A) but not observed in the liver (FIG. 2B). On the other hand,after the macrophages were injected, fluorescent cells were hardlyobserved in normal brain cells (FIG. 2C), while many fluorescent cellswere observed in the liver (FIG. 2D).

Accordingly, the established cell line of microglia of the presentinvention possessed specific affinity for the brain.

(b-2) Functional Characteristics (ability to produce IL-1 and IL-6)

1×10⁶ Ra2 cells were plated onto a 6 cm culture vessel, and total RNAwas extracted from the cells stimulated with lipopolysaccharides for 12hours and from the cells not stimulated by using RNeasy (Qiagen Co.),and 2 μg of the RNA was used to prepare a cDNA mixture by reversetranscriptase (BRL). PCR was carried out using the resulting cDNA as atemplate, where an IL-1 specific synthetic primer and IL-6 specificsynthetic primer having the following sequences were used. IL-1 specificsynthetic primer (Sawada et al., Int. J. Dev. Neurosci, 13, 253-264,1995): (SEQ ID NO:1) Sense chain: 5′-ATGGCAACTGTTCCTGAACTCAACT-3′ (SEQID NO:2) Antisense chain: 5′-CAGGACAGGTATAGATTCTTTCCTTT-3′

IL-6 specific synthetic primer (Sawada et al., Brain Res. 583, 296-299,1992): (SEQ ID NO:3) Sense chain: 5′-ATGAAGTTCCTCTCTGCAAGAGACT-3′ (SEQID NO:4) Antisense chain: 5′-CACTAGGTTTGCCGAGTAGATCTC-3′

In PCR, 30 cycles each consisting of reaction at 55° C. for 1 minute,72° C. for 2 minutes and 94° C. for 1 minute were carried out (Omnigenefrom HYBAID Co., Ltd. was used). After PCR, the amplification productwas subjected to agarose gel electrophoresis to examine gene expression.

As a result, it was found that Ra2 increased expression of IL-1 and IL-6(FIG. 3, “LPS” lanes). In FIG. 3, “M” is a molecular weight marker and“cont” is control (not stimulated).

Production of IL-1 and IL-6 was also confirmed by ELISA. Further, aculture supernatant of the established microglia of the presentinvention after stimulation with lipopolysaccharides was added to MH60cells proliferating depending on IL-6 or to D10 cells proliferatingdepending on IL-1, followed by incubation, and whether the MH60 cellsand D10 cells were proliferated or not was examined.

As a result, it was found that both the cells proliferated in thepresence of the culture supernatant of the established microglia of thepresent invention stimulated with the lipopolysaccharides.

(c) Cell Growth Ability

5×10⁴ GMI-R1 cells were plated on a 96-well test plate, and 2 μg/mlrGM-CSF was added to it so as to be diluted 1000-, 5000- and 10000-foldrespectively, and the cells were incubated for 4 days and then subjectedto MTT assays. As a control, 400 μ/ml human M-CSF (The Green CrossCorporation) was diluted 1000-fold and 0.1 mg/ml PMA (phorbol myristateacetate) was diluted 1000- or 5000-fold, and these were used as thecontrol (FIG. 4A).

Separately, rGM-CSF diluted 5000-fold, and 100 μ/ml each of mouse IL-3,IL-4 and IL-6 (any of which were produced by Genzyme Co.), were examinedin a comparative test. Two days and four days after addition of therespective reagents, MTT assays were carried out (FIG. 4B).

As a result, it was found that GMI-R1 was proliferated depending onrGM-CSF.

Example 2

Introduction of a Gene into the Microglia of the Present Invention

Vector ptkβ (Clonetech Co.) for expression of lac Z gene derived from E.coli and DOTAP lipid (Boehringer-Mannheim Co.) were mixed to be a finalconcentration of 1 μg/ml. The mixture was mixed with a serum-containingmedium, then added to the established cell line of microglia of thepresent invention and treated for 16 hours. As the control, theestablished microglia of the invention into which the gene was notintroduced, and macrophages obtained in the same manner as in Example 1,were used.

Then, the cells were further cultured for 48 hours in a usual medium(EMEM plus 10% FCS) and then stained with the fluorescent pigment asdescribed in Example 1 (b-1) in order to examine whether the gene wasdelivered to and expressed in the brain, as follows: An artery in theleft armpit of a mature rat (250 to 300 g) under anesthesia withNembutal was exposed. After hemostatic treatment, a cannula was insertedinto the artery and used to inject 1 to 2×10⁶ cells into the rat. Afterinjection, the incision site was sutured and the rat was allowed torecover.

48 hours after the cells were injected, the brain was excised from therat and 3 successive frozen sections of the brain were prepared andobserved respectively under a fluorescence microscope. Further, stainingand quantification of β-galactosidase activity was carried out in thefollowing manner.

One of the three sections was fixed in 0.5% glutaraldehyde and subjectedto activity staining with Xgal as substrate according to the method ofLim et al. (Bio Techniques7, 576-579, 1989). To quantify the activity,one of the sections was homogenized in a dissolving buffer byultrasonication and examined for its activity with a commercial kit(Galacto Light; Boehringer-Mannheim).

As a result, it could be confirmed that in case the gene was introducedinto the established microglia of the present invention, lacZ-positivecells were present in the section from the rat brain (FIG. 5). Further,as a result of quantification of β-galactosidase activity by thechemiluminescence method, considerably higher activity was detected inthe rat brain section into which the E. coli derived gene lac Zexpression vector was introduced than the counterpart into which thegene was not introduced (FIG. 6).

Example 3

Introduction of Chemical Substance into the Microglia of the Invention,and Specific Introduction Thereof into the Brain

The fluorescent pigment PKH26 used in Example 1 forms granules indiluent B. The microglia cell strain of the present inventionincorporates these granules specifically and transfers them to thebrain, so the fluorescent pigment PKH26 was used as a model of chemicalsubstance (anti-tumor drug).

As a result, when the established cell line of microglia of the presentinvention was introduced, many fluorescent cells were observed in normalbrain cells but not observed in the liver. It therefore follows that themicroglia of the present invention transfers the chemical substance(drug) into the brain specifically.

Example 4

Introduction of Gene with GFP

1×10⁶ GMI-R1 cells were plated on a Petri dish, and 16 hours later, 10μg GFP expression vector pEGFP (Clonteck Co.) was added to agene-introducing medium, with which the previous medium was thenexchanged, and the cells were cultured at 37 ° C. for 24 hours in a CO₂incubator and then cultured together with rGM-CSF for additional 7 daysin a medium for microglia.

After culture for 7 days, the cells were suspended, and those cellsexhibiting fluorescence intensity which was at least 100 times as highas that of the cells into which the gene had not been introduced werefractionated and concentrated by a fluorescent activated cell sorter(FACS), FACS Calibur produced by Becton-Dickinson and then culturedtogether with rGM-CSF in a medium for microglia.

The same procedure was repeated additionally twice every 7 days, wherebyalmost 90% or more cells could be recovered in a fraction having about100-fold fluorescence, and these cells were put to a TP96 test plate ata density of 1 cell/well by limiting dilution of the cells and furthercultured together with rGM-CSF in a medium for microglia. The microgliacoming to expressing the introduced gene pEGFP stably and constantlycould thereby be separated.

One example of results in FACS analysis as to how the cells could beseparated by this method is shown in the drawings. FIG. 7 shows a resultof FACS analysis of the cells into which the gene was not introduced,while FIG. 8 shows a result of FACS analysis of the cells into which GFPwas introduced.

Industrial Applicability

According to the present invention, there is provided an establishedcell line of microglia having specific affinity for the brain. Theestablished cell line of microglia according to the present invention isuseful not only as a carrier for introducing a gene into the brain, butalso as a carrier for introducing a chemical substance such as drugspecifically into the brain.

1-16. (canceled)
 17. A method for the production of establishedmicroglia cell lines having specific affinity for the brain, whichcomprises the steps of: a) separating and purifying type-II microgliacells from microglia cells isolated from animals, and b) incubating thetype II microglia cells with cytokines.
 18. The method according toclaim 17, wherein the type II microglia cells are separated and isolatedby incubating microglia cells isolated from animals in an incubationvessel having no surface charge.
 19. The method according to claim 18,wherein the incubation vessel having no surface charge is a non-coatedplastic vessel.
 20. The method according to claim 17, wherein thecytokines are selected from colony-stimulating factor (CSF),granulocyto-macrophage colony-stimulating factor (GM-CSF), or acombination thereof.
 21. The method according to claim 20, wherein thecytokines consist of colony-stimulating factor (CSF).
 22. The methodaccording to claim 20, wherein the cytokines are granulocyto-macrophagecolony-stimulating factor (GM-CSF).
 23. The method according to claim20, wherein the cytokines are recombinant cytokines.